Toxicity
To the eyes of an expert in the art, and bearing in mind the knowledge in the state of the art, the toxic characteristics of crotamine may inhibit studies aiming its use for any beneficial purpose for human beings and other living organisms.
Crotamine is a toxin, more specifically a myotoxin, isolated from the venom of South American rattlesnake, Crotalus durissus terrificus. This toxin is one of the most abundant component in the venom of rattlesnake, corresponding to approximately 10% of dry weight of the crude venom. Crotamine is a basic polypeptide with a low molecular weight of about 4,800 Daltons, with isoelectric point above 9.5. It is constituted by 42 amino acid residues (YKQCHKKGGHCFPKEKICLPPSSDFGKMDCRWRWK CCKKGSG (SEQ ID NO: 2)), showing six cysteine residues forming three disulphide bridges, and it is rich in basic amino acids, such as lysine and arginine.
When injected intraperitoneally, crotamine causes a quick paralysis, in less than 15 minutes, of the hind legs of mice, which is the typical physiological effect of this toxin. Furthermore, difficulty in breathing and rigidity are also observed, suggesting veratrine-simile action (Gonçalves, J. M. (1956), as mentioned above. At cellular and molecular levels, crotamine induces an increase in the voltage-dependent sodium current (as mediated by sodium channels) by causing high depolarization (reduction of rest potential) of the membrane of myocites of muscle fibers next to motor plates—a mechanism which is prevented by tetradotoxin (TTX). Consequently, the massive inflow of sodium ions causes dilatation of the sarcoplasmatic reticulum of myocites and the slow induction of myonecrosis restricted to the cells of skeletal muscles.
This mechanism, through which crotamine exerts its toxicity, is similar to that described for myotoxin-a from Crotalus viridis viridis, a more extensively studied toxin.
Crotamine is able to form dimers through linkages between disulphide chains, acquiring a form close to a sphere, which covalently links extra subunits.
Therefore, there are no publications in the state of the art suggesting or indicating, in any form, benefits incurred from the effects of crotamine on living organisms.
Cell Penetration
Cell-penetrating peptides (CPP, or “cell-translocating peptides”) are known in the state of the art. A few characteristics of this type of peptide were already published, such as its cationic character, low molecular weight and high content of basic amino acids. But these peptides may vary in their primary structure, size and/or ability to penetrate cells, which makes it difficult to recognize a CPP. The basic structural characteristic leading to the cell penetration feature is still unknown. Some natural CPPs have been described, for instance, the protein HIV-1 TAT, derived from the human immunodeficiency virus (HIV-1) and isolated from the virus transcription activation factor, it shows the ability to penetrate into cells in vitro, with a cytoplasmic and nuclear localization; Antp43-58, derived from the transcription factor of the Antennapedia homeodomain from Drosophila; and also the structural protein of Herpes simplex virus type 1 (HSV-1), named as VP22. These three cell-penetrating proteins have protein transduction domains (PTDs), i.e., a sequence of amino acid residues that in the isolated form presents a higher cell penetration efficiency.
However, even considering the few standard common structural characteristics of a cell-penetrating peptide, a skilled man in the art would not be able to predict the effect of the presence of the three disulphide bonds of crotamine in the cell-penetrating property. These disulphide bonds are formed by six cysteines present in crotamine molecule, and is one of the structural characteristics that distinguishes it from other known natural cell-penetrating peptides.
Transport of Molecules to Cytoplasm and Cell Nucleus
In the state of the art, few technologies on the transport of genetic material to the cytoplasm and/or cell nucleus are known, but they all present a few shortcomings, such as the lack of cell selectivity and low efficiency of molecule loading. The use of cationic peptides isolated from snake venom for these purposes, such as crotamine, has never been published before.
References covering the subject, as generically associated to this aspect of the invention (intracellular and nuclear penetration and/or DNA and/or molecule transport), are all of them different from the present invention and none of them indicate nor suggest in any form, any feature of the crotamine, as described by the Applicant herein.
The U.S. Pat. No. 4,774,318 discloses the isolation of a small polypeptide with cytotoxic activity, purified from the low molecular weight fraction of the rattlesnake Crotalus atrox. This compound may be used as an inhibitor for cell growth, both pure and in combination with other reagents, such as antibodies. Conjugates of these peptides with specific binding members, e.g., ligands and receptors, may be used for selectively removing cells from a mixture of cells.
The U.S. Pat. No. 6,420,176 discloses a composition for DNA transfer to antigen presenting cells. A molecular complex for a specific transfer to antigen presenting cell is formed by a non-viral gene transfer system complexed with a foreign genetic material. The complex then enters the target cell through specific receptors and shows resistance to the intracellular degradation mechanisms. Consequently, the incorporation or transduction of said foreign genetic material results in the expression of the corresponding protein. The patent also includes a gene immunization method without using needles.
The U.S. Pat. No. 6,461,641 discloses a delivery techniques of therapeutic reagents for tumor treatment. The main problem of usual methods for the delivery of therapeutic reagents, into solid tumors, specially of cells or large volumes of recombinant DNA reagents or drugs, has been the resistance of said tissues to the influx of cells and/or fluids, resulting in a low efficiency of the fluid and/or cell penetrating into and remaining in the tumor tissues to be treated. The use of more viscous vehicle, preferably with similar density of the tissue, allows more efficient penetration and reduced backflow and diversion through the point of entry, so that more material may be introduced and remains in the tumor. Preferred materials include solutions or suspensions of a polymeric material which gel or solidify at the time of or shortly after injection or implantation.
The U.S. Pat. No. 6,635,623 discloses lipoprotein as a vector for the transport of nucleic acids. This invention relates to the materials and methods for the transport and conduction of nucleic acids in vivo. It specifically refers to the use of lipoproteins, including low density lipoproteins (LDL) and/or as apolipoproteins, to bind and transport nucleic acids in vivo. Furthermore, this invention refers to the use of lipoproteins for the early detection of cancer and/or metastasis and/or arteriosclerosis.
The U.S. Pat. No. 6,638,767 discloses methods to deliver compounds into the cells by using organic halides and/or carriers, which may be associated with the use of ultrasound. Organic halide is defined as a halogenated organic compound, i.e., containing at least one carbon atom and at least one halogen atom which may be fluorine (preferably), chlorine, bromine or iodine.
The U.S. Pat. No. 6,676,935 discloses tissue-specific adenovirus as a deliver agent for transfecting target host cell. By providing for transcriptional initiating regulation dependent upon transcription factors which are only active for specific and restricted cell types, viral replication may be restricted to target cells. Modified adenovirus may be used as a carrier for introducing the genetic material, particularly associated with cytotoxicity for treating neoplasia.
The U.S. Pat. No. 6,680,301 discloses a method for transferring molecules to cells by disrupting endosomal and lysosomal membranes using photodynamic treatment, without killing the majority of the cells by the photodynamic treatment. More specifically, this invention includes a DNA and/or RNA transfer method, such as genes, to cells by photochemically inducing the disruption of endosomes and lysosomes.
The U.S. Pat. No. 6,692,911 discloses a composition to deliver compounds inside cells and specifically refers to biocompatible endosomolytic agents. In an ideal formulation, endosomolytic agents are biodegradable and may disintegrate within cells into compounds which may be re-used or released by the cell. Endosomolytic agents include cationic polymers, specifically compounds constituted by biomolecules, such as histidine, poly-histidine, poly-lysine or any combination thereof. Another example of an endosmolytic agent includes imidazole-containing compounds such as vinylimidazole and histamine. Agents having multiple proton acceptor sites and acting as a “proton sponge”, disrupting the endosome by osmolytic action. In preferred embodiments, this patent also contemplates the use of these endosomolytic agents as delivery agents by complexation with the desired compound to be delivered.
The known methods used for intracellular DNA delivery may be basically classified as viral and non-viral systems. It should be considered that the efficiency in DNA transport depends on steps such as: the absorption of the transfection complex in contact with the cell surface, internalization of said complex by the cell, release from the endosomes and translocation to the nucleus for gene expression. Viral carriers are able to overcome all these obstacles, since they express proteins that facilitates the gene transportation through these several levels. However, these vectors present limited DNA transport ability, besides several shortcomings, such as, problems in the packaging and production process, risk of viral recombination, toxicity and immunogenicity in vivo. On the other hand, although the non-viral carriers appear as powerful tools to elucidate gene function and regulation, they present low efficiency for DNA delivery to the nucleus of the target cells. The non-viral system is safer than the viral system and it has been currently used for clinical tests, but its low efficiency in the transgene expression remains to be its main disadvantage. However, crotamine, as a non-viral carrier, as described in the present invention, shows a surprisingly effective DNA delivery to the cytoplasm and/or to the cell nucleus in vitro and mainly in vivo.
Therefore, the uses of crotamine, as described in the present invention, could not be foreseen so far, and they have never been disclosed or suggested in the state of the art, including the lack of toxicity at lower concentrations, the ability to penetrate cells or transport of genetic material or other molecules to the surface, cytoplasm or cell nucleus, besides its surprising selectivity for actively proliferating cells.